RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions

Abstract

Prokaryotes have evolved multiple versions of an RNA-guided adaptive immune system that targets foreign nucleic acids. In each case, transcripts derived from clustered regularly interspaced short palindromic repeats (CRISPRs) are thought to selectively target invading phage and plasmids in a sequence-specific process involving a variable cassette of CRISPR-associated ( cas ) genes. The CRISPR locus in Pseudomonas aeruginosa (PA14) includes four cas genes that are unique to and conserved in microorganisms harboring the Csy-type (CRISPR system yersinia) immune system. Here we show that the Csy proteins (Csy1–4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences. Target recognition is enthalpically driven and localized to a “seed sequence” at the 5′ end of the CRISPR RNA spacer. Structural analysis of the complex by small-angle X-ray scattering and single particle electron microscopy reveals a crescent-shaped particle that bears striking resemblance to the architecture of a large CRISPR-associated complex from Escherichia coli , termed Cascade. Although similarity between these two complexes is not evident at the sequence level, their unequal subunit stoichiometry and quaternary architecture reveal conserved structural features that may be common among diverse CRISPR-mediated defense systems.

Type I CRISPR-Cas targets endogenous genes and regulates virulence to evade mammalian host immunity

“…Csy1-4 proteins constitute the cascade [32], wherein Csy4 func-tions in processing crRNA transcripts, whereas Csy1-3 proteins are required to stabilize the crRNA produced by Csy4 [32]. On the other hand, Cas1 is not required for this procedure, but has a role in acquiring new spacers [33].…”

Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition

“…Both Cas9 [26,27] and Cpf1 [19,24] have been shown to have a seed sequence at the PAM-proximal side of the protospacer, which is critical for DNA recognition and cleavage. The 10-nt seed sequence of the guide RNA has been shown to form a preordered A-form conformation in the Cas9-sgRNA complex to facilitate guide-target duplex formation [14], a mechanism that has also been found in eukaryotic Argonaute complexes [28][29][30].…”

CRISPR control of virulence in Pseudomonas aeruginosa

“…In P. aeruginosa, numerous spacers in the CRISPR loci are complementary to sequences in bacteriophage genomes [5,6]. While some of the spacers are perfectly complementary, others contain several mismatches.…”

“…The complementary target sequence is referred to as a protospacer (i.e., origin of the spacer) and the consecutive GC base pairs are referred to as the Protospacer Adjacent Motif (PAM). Previous DNA binding studies have shown that the PAM and DNA target base-pairing with the first 8 bases of the crRNA, the so-called "seed" region, are critical for high-affinity binding [6]. Csy complex binding to a target with the correct PAM, base pairing within the seed, and sufficient base pairing throughout the remainder of the protospacer, trigger recruitment of a trans acting nucleasehelicase protein called Cas3 that degrades the target DNA.…”

“…In P. aeruginosa, each crRNA assembles with four different Cas proteins (i.e., Csy 1-4) to form a surveillance complex, called the Csy complex or Cascade I-F (Figure 1) [6]. The Csy complex binds to foreign DNA in sequential steps, initiated by detection of two consecutive guanine-cytosine base pairs (G-C/G-C) located adjacent to the complementary DNA target [10].…”